Improvement of microorganisms of the genus Corynebacterium or Brevibacterium by recombinant DNA technology has been attempted using host-vector systems for these genera. However, recombinant plasmids introduced into a host microorganism are often unstable and are liable to be lost from the host. Therefore, the instability of a recombinant plasmid is one of the serious problems to be solved for practical use of the microorganisms constructed by recombinant DNA technology in an industrial process for producing amino acids.
So far, several methods to stabilize plasmids in host cells have been reported in microorganisms of genera other than Corynebacterium and Brevibacterium. These methods are classified into two types according to the manner of stabilization. First, genetic stabilization of a plasmid is known. For example, as for Escherichia coli, it is known that the par gene which relates to stable distribution of the low copy number plasmid pSC101 is inserted into the plasmid [Gene, 23, 105 (1983)]. Likewise, as for strains of the genus Corynebacterium or Brevibacterium, it is known stabilize a plasmid capable of autonomous replication in Coryneform bacteria of the genus Brevibacterium in such bacteria by insertion of a gene which is present on a plasmid carried by Brevibacterium stationis (EP-352763A1). Such methods afford improvement in stability of a plasmid to some extent, but are not suitable for an industrial process for amino acid production involving several-steps of seed culturing because it is known that plasmids are liable to be lost from host cells through repetitions of cell division for proliferation during cultivation [Bio/Technology, 4, 901 (1986)]. Second, methods for selectively growing plasmid-carrying cells by inhibiting the growth of cells from which a plasmid has been lost are known. For example, in small scale cultivation, antibiotics are usually added to the culture medium. However, such addition of antibiotics is costly in large scale cultivation. Further, the purification step for removal of the added antibiotics is required, and thus this method is not suitable for an industrial process. As a method without such addition of specific substances, it is known to stabilize plasmid in host cells by using a combination of a host strain which has deficiency in a gene essential for its growth and a plasmid containing a gene which complements the deficiency. For example, for Bacillus subtilis, a combination of a host strain which is deficient in the alanine racemase gene and a plasmid containing the alanine racemase gene of which type is employed [Bio/Technology, 3, 1003 (1985)]; and for Escherichia coli, a combination of a diaminopimelic acid-requiring host strain and a plasmid containing a gene which complements the requirement is employed (Japanese Published Unexamined Patent Application No. 233790/88). In both cases, plasmid-free cells are lysed because of their incapability of synthesizing D-alanine or diaminopimelic acid which is essential component of their cell walls, and thus only cells carrying the plasmids can grow normally. These methods are applicable to a cultivation processes using culture media which contain extracts derived from animals, plants or yeasts and which are usually used in industrial fermentation processes, as such natural nutrients do not contain D-alanine or diaminopimelic acid.
However, it is difficult to apply these methods to microorganisms which have isozymes of alanine racemase or have a bypass of diaminopimelic acid biosynthetic pathway because D-alanine or diaminopimelic acid-requiring mutants of such microorganisms can not be readily obtained.
In the large scale industrial production of substances such as amino acids by using strains of the genus Corynebacterium or Brevibacterium carrying recombinant plasmids, cells proliferate by repetitions of division through steps from seed culture to fermentation for the production, during which the recombinant plasmids are often lost from the cells. This instability of the plasmids causes a reduction of productivity, and therefore, it is desired to develop a method to stabilize recombinant plasmids in the host cells, leading to the industrial production of substances at low cost.
There is no report on the stabilization of a plasmid in a host strain by the use of a mutant having requirement for substances contained in natural nutrients added as medium components, e.g. amino acids, as the host, and a plasmid containing a gene which complements the requirement of the host as the vector. Such a combination has been considered to be inappropriate as a host-vector system for stabilizing a plasmid in a host strain because cells from which plasmids have been lost can also grow.